小麦、水稻、玉米、白菜、芹菜内生固氮菌及其系统发育

doi:10.3864/j.issn.0578-1752.2012.07.008

摘要/Abstract

摘要： 【目的】了解小麦、水稻、玉米、白菜、芹菜内生固氮菌的主要类群，确定内生固氮菌的系统发育地位。【方法】样品表面灭菌后采用无氮培养基分离、培养内生固氮菌，乙炔还原法测定菌株固氮酶活性；PCR扩增得到菌株16S rDNA，通过序列测定和相似性分析研究菌株的系统发育。【结果】从大田小麦体内分离到内生固氮菌34株，固氮酶活性在0.30—30.24 nmol C2H4/h•mg蛋白，基于16S rDNA序列最大相似性，这些菌株分属于假单胞菌（Pseudomonas）、根瘤菌（Rhizobium）、芽孢杆菌（Bacillus）、黄杆菌（Flavobacterium）等13属21种，种群分布较为广泛；从大田水稻体内分离到内生固氮菌25株，固氮酶活性在3.12—254.12 nmol C2H4/h•mg蛋白，属于芽孢杆菌、伯克霍尔德氏菌（Burkholderia）、肠杆菌（Enterobacter）、克雷伯氏菌（Klebsiella）等9属16种，伯克霍尔德氏菌、肠杆菌和克雷伯氏菌是水稻内生固氮菌特有种群；从大田玉米体内分离到内生固氮菌9株，固氮酶活性在7.27—59.58 nmol C2H4/h•mg蛋白，属于根瘤菌、鞘氨醇单胞菌（Sphingomonas）等5属6种；从盆栽试验小白菜体内分离到内生固氮菌14株，固氮酶活性在2.33—205.21 nmol C2H4/h•mg蛋白，属于根瘤菌、节杆菌（Arthrobacter）等6属8种；从市售芹菜体内分离到内生固氮菌10株，固氮酶活性在1.23—46.70 nmol C2H4/h•mg蛋白，属于鞘氨醇单胞菌、假单胞菌等5属8种。【结论】在生长期小麦、水稻、玉米和部分蔬菜体内普遍存在内生固氮菌，菌株固氮酶活性差异较大，在0.30—254.12 nmol C2H4/h•mg蛋白，系统发育地位分属于假单胞菌、根瘤菌、芽孢杆菌等25个属的56个种，这些内生固氮菌对于农业生产有巨大潜能。

关键词: 小麦, 水稻, 玉米, 白菜, 芹菜, 内生固氮菌, 系统发育

Abstract: 【Objective】 The objective of this study is to determine the main groups and phylogenetic position of nitrogen-fixing endophytic bacteria in wheat, rice, maize, Chinese cabbage and celery. 【Method】 Surface sterilization and nitrogen-free medium were used to isolate nitrogen-fixing endophytic bacteria. Acetylene reduction assay was used to measure the nitrogenase activity. 16S rDNA was amplified by PCR and 16S rDNA sequence was analysed with MEGA software. 【Result】 Thirty four nitrogen-fixing endophytic bacteria with nitrogenase activity ranging from 0.30 to 30.24 nmol C2H4/h•mg protein were isolated from field wheat. Based on 16S rDNA sequence similarity, these strains belonged to 21 species of 13 genera, including Pseudomonas, Rhizobium, Bacillus, Flavobacterium, etc. Twenty five nitrogen-fixing endophytic bacteria with nitrogenase activity ranging from 3.12 to 254.12 nmol C2H4/h•mg protein were isolated from field rice. They belonged to 16 species of 9 genera, including Bacillus, Burkholderia, Enterobacter, Klebsiella, etc. Moreover, Burkholderia, Enterobacter and Klebsiella were dominent. Nine maize endophytic diazotrophs with nitrogenase activity ranging from 7.27 to 59.58 nmol C2H4/h•mg protein were identified as 6 species of genera Rhizobium, Sphingomonas, Arthrobacter, Brevibacterium and Microbacterium. Fourteen endophytic diazotrophs with nitrogenase activity ranging from 2.33 to 205.21 nmol C2H4/h•mg protein were isolated from pot trial pakchoi and identified as 8 species of 6 genera of Rhizobium, Arthrobacter, Pseudomonas, etc. Ten endophytic diazotrophs with nitrogenase activity ranging from 1.23 to 46.70 nmol C2H4/h•mg protein were isolated from marcket celery and identified as 8 species of 5 genera of Sphingomonas, Pseudomonas, Brevundimonas, etc. 【Conclusion】 Nitrogen-fixing endophytic bacteria colonized widely in wheat, rice, maize and vegetables. Their nitrogenase activity under pure culture ranged from 0.30 to 254.12 nmol C2H4/h•mg protein. Ninety two endophytic diazotrophs belonged to 56 species of 25 genera, including Pseudomonas, Rhizobium, Bacillus, Burkholderia, Sphingomonas, Enterobacter and Klebsiella, etc. These endophytic diazotrophs have a great potential to agriculture.

Key words: wheat, rice, maize, Chinese cabbage, celery, endophytic diazotrophs, phylogeny

孙建光, 罗琼, 高淼, 胡海燕, 徐晶, 周义清. 小麦、水稻、玉米、白菜、芹菜内生固氮菌及其系统发育[J]. 中国农业科学, 2012, 45(7): 1303-1317.

SUN Jian-Guang, LUO Qiong, GAO Miao, HU Hai-Yan, XU Jing, ZHOU Yi-Qing. Isolation and Phylogeny of Nitrogen-Fixing Endophytic Bacteria in Wheat, Rice, Maize, Chinese Cabbage and Celery[J]. Scientia Agricultura Sinica, 2012, 45(7): 1303-1317.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2012.07.008

https://www.chinaagrisci.com/CN/Y2012/V45/I7/1303

参考文献

[1]Barraquio W, Revilla L, Ladha J K. Isolation of endophytic diazotrophic bacteria from wetland rice. Plant and Soil, 1997, 194: 15-24.

[2]Zhang G X, Peng G X, Wang E T, Yan H, Yuan Q H, Zhang W, Lou X, Wu H, Tan Z Y. Diverse endophytic nitrogen-fixing bacteria isolated from wild rice Oryza rufipogon and description of Phytobacter diazotrophicus gen. nov. sp. nov.. Archieve Microbiology, 2008, 189: 431-439.

[3]Peng G, Zhang W, Luo H, Xie H, Lai W, Tan Z. Enterobacter oryzae sp. nov., a nitrogen-fixing bacterium isolated from the wild rice species Oryza latifolia. International Journal of Systematic and Evolutionary Microbiology, 2009, 59: 1650-1655.

[4]Sandhiya G S, Sugitha T C, Balachandar D, Kumar K. Endophytic colonization and in planta nitrogen fixation by a diazotrophic Serratia sp. in rice. Indian Journal of Experimental Biology, 2005, 43(9): 802-807.

[5]Govindarajan M, Balandreau J, Kwon S W, Weon H Y, Lakshminarasimhan C. Effects of the inoculation of Burkholderia vietnamensis and related endophytic diazotrophic bacteria on grain yield of rice. Microbial Ecology, 2008, 55(1): 21-37.

[6]Baldani V L D, Baldani J I, Döbereiner J. Inoculation of rice plants with the endophytic diazotrophs Herbaspirillum seropedicae and Burkholderia spp.. Biology and Fertility Soils, 2000, 30: 485-491.

[7]Rodrigues E P, Rodrigues L S, de Oliveira A L M, Baldani V L D, dos Santos Teixeira K R, Urquiaga S, Reis V M. Azospirillum amazonense inoculation: effects on growth, yield and N2 fixation of rice (Oryza sativa L.). Plant Soil, 2008, 302: 249-261.

[8]Estrada-De los Santos P, Bustillos-Cristales R, Caballero-Mellado J. Burkholderia, a genus rich in plant-associated nitrogen fixers with wide environmental and geographic distribution. Applied and Environmental Microbiology, 2001, 67(6): 2790-2798.

[9]Estrada P, Mavingui P, Cournoyer B, Fontaine F, Balandreau J, Caballero-Mellado J. A N2-fixing endophytic Burkholderia sp. associated with maize plants cultivated in Mexico. Canidian Journal of Microbiology, 2002, 48(4): 285-294.

[10]Boddey R M, Urquiaga S, Alves B J R, Reis V. Endophytic nitrogen fixation in sugarcane: present knowledge and future applications. Plant and Soil, 2003, 252: 139-149.

[11]Govindarajan M, Kwon S W, Weon H Y. Isolation, molecular characterization and growth-promoting activities of endophytic sugarcane diazotroph Klebsiella sp. GR9. World Journal of Microbiology and Biotechnology, 2007, 23: 997-1006.

[12]dos Reis Junior F B, Reis V M, Urquiaga S, Döbereiner J. Influence of nitrogen fertilisation on the population of diazotrophic bacteria Herbaspirillum spp. and Acetobacter diazotrophicus in sugar cane (Saccharum spp.). Plant and Soil, 2000, 219: 153-159.

[13]Oliveira A L M, Urquiaga S, Döbereiner J, Baldani J I. The effect of inoculating endophytic N2-fixing bacteria on micropropagated sugarcane plants. Plant and Soil, 2002, 242: 205-215.

[14]Govindarajan M, Balandreau J, Muthukumarasamy R, Revathi G, Lakshminarasimhan C. Improved yield of micropropagated sugarcane following inoculation by endophytic Burkholderia vietnamiensis. Plant and Soil, 2006, 280: 239-252.

[15]李倍金, 罗明, 周俊, 孔德江, 张铁明. 几种禾草内生固氮菌的分离及固氮活性测定. 草业学报, 2008, 17(5): 37-42.

Li B J, Luo M, Zhou J, Kong D J, Zhang T M. Isolation of endophytic diazotrophic bacteria from several gramineae grasses and determination of their nitrogenase activity. Acta Prataculturae Sinica, 2008, 17(5): 37-42. (in Chinese)

[16]Tan Z Y, Peng G X, Xu P Z, Ai S Y, Tang S H, Zhang G X, Zeng F Y. Diversity and high nitrogenase activity of endophytic diazotrophs isolated from Oryza rufipogon Griff.. Chinese Science Bulletin, 2009, 54: 2839-2848.

[17]孙建光, 张燕春, 徐晶, 胡海燕. 高效固氮芽孢杆菌选育及其生物学特性研究. 中国农业科学, 2009, 42(6): 2043-2051.

Sun J G, Zhang Y C, Xu J, Hu H Y. Isolation and biological characteristic investigation on efficient nitrogen-fixing bacilli. Scientia Agricultura Sinica, 2009, 42(6): 2043-2051. (in Chinese)

[18]孙建光, 徐晶, 胡海燕, 张燕春, 刘君, 王文博, 孙燕华. 中国十三省市土壤中非共生固氮微生物菌种资源研究. 植物营养与肥料学报, 2009, 15(6): 1450-1465.

Sun J G, Xu J, Hu H Y, Zhang Y C, Liu J, Wang W B, Sun Y H. Collection and investigation on asymbiotic nitrogen-fixing microbial resources from 13 provinces over China. Plant Nutrition and Fertilizer Science, 2009, 15(6): 1450-1465. (in Chinese)

[19]Franche C, Linderstrom K, Elmerich C. Nitrogen-fixing bacteria associated with leguminous and non-leguminous plants. Plant Soil, 2009, 321: 35-59.

[20]Muthukumarasamy R, Cleenwerck I, Revathi G, Vadivelu M, Janssens D, Hoste B, Gum K U, Park K D, Son C Y, Sa T, Caballero-Melladoe J. Natural association of Gluconacetobacter diazotrophicus and diazotrophic Acetobacter peroxydans with wetland rice. Systematic and Applied Microbiology, 2005, 28: 277-286.

[21]Jha B, Thakura M C, Gontia I, Albrecht V, Stoffels M, Schmid M, Hartmann A. Isolation, partial identification and application of diazotrophic rhizobacteria from traditional Indian rice cultivars. European Journal of Soil Biology, 2009, 45: 62-72.

[22]Prakamhang J, Minamisawa K, Teamtaisong K, Boonkerd N, Teaumroong N. The communities of endophytic diazotrophic bacteria in cultivated rice (Oryza sativa L.). Applied Soil Ecology, 2009, 42: 141-149.